The ecosystem carbon (C) balance in permafrost regions, which has a global significance in understanding the terrestrial C-climate feedback, is significantly regulated by nitrogen (N) dynamics. However, our knowledge on temporal changes in vegetation N limitation (i.e., the supply of N relative to plant N demand) in permafrost ecosystems is still limited. Based on the combination of isotopic observations derived from a re-sampling campaign along a ~3000 km transect and simulations obtained from a process-based biogeochemical model, here we detect changes in ecosystem N cycle across the Tibetan alpine permafrost region over the past decade. We find that vegetation N limitation becomes stronger despite the increased available N production. The enhanced N limitation on vegetation growth is driven by the joint effects of elevated plant N demand and gaseous N loss. These findings suggest that N would constrain the future trajectory of ecosystem C cycle in this alpine permafrost region.